Throughout this application, various publications are referenced in parentheses by author and year. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
The need to study many biological targets simultaneously drives the development of multiplex fluorescent tags. However, due to the limits of the spectral region, and therefore the availability of appropriate detectors, the number of available fluorescent dyes that have distinguishable emission spectra is limited to about ten. To overcome this limitation, a combinatorial fluorescent labeling approach for multi-color fluorescence in situ hybridization (M-FISH) has been developed and is now widely used in the field of cytogenetics (Speicher et al., 1996; Schrock et al., 1996). This approach mixes from two to seven individual fluorescent dyes that have unique emissions, and uses the fluorescence emission pattern to identify the different targets. The unique fluorescence emission pattern is achieved by mathematically combining the different dyes. This development has made possible advances in chromosome analyses. However, the procedure requires physically mixing the individual dyes in a quantitative manner to develop xe2x80x9cuniquexe2x80x9d probe labels. This requirement, coupled with the potential interactions of the dyes, complicates the fluorescence emission patterns. Therefore, the major application of the technique is limited to methods that involve hybridization. Multiple lasers and detectors are also required for the imaging. A reagent kit that can be used to covalently label a wide range of biomolecules is difficult to construct with this approach. Thus, there is an urgent need for a large set of fluorescent tags that can be used for multiple component analyses in biomedical and other fields. Previously, the principle of fluorescent energy transfer (ET) was used to enhance fluorescence emission for the successful development of four ET tags for deoxyribonucleic acid (DNA) sequencing which are widely used in the Human Genome Project (Ju et al. 1995, 1996).
The present application discloses how energy transfer and combinatorial concepts can be used to tune the fluorescence emission signatures of the fluorescent tags for the development of a large number of combinatorial fluorescence energy transfer (CFET) tags. All the CFET tags can be excited with a single laser source and analyzed by simple detectors. Such CFET tags should be valuable for multiplex genetic mutation analysis, DNA mapping, and genome-wide chromosome analysis, as well as for other multi-component analysis systems.
This invention is directed to a combinatorial fluorescence energy transfer tag which comprises a plurality of fluorescent molecules, comprising one or more energy transfer donor and one or more energy transfer acceptor, linked through a molecular scaffold wherein the fluorescent molecules are separated along the scaffold to produce a unique fluorescence emission signature.
The invention provides a combinatorial fluorescence energy transfer tag which comprises the structure: 
wherein A, B, and C represent different fluorescent molecules which comprise one or more energy transfer donor and one or more energy transfer acceptor, wherein m and n represent any integer greater than or equal to one, and wherein fluorescent molecules A, B, and C are separated from each other to produce a unique fluorescence emission signature.
The invention provides oligonucleotide primers, oligonucleotide probes, nucleotides, deoxynucleosides, and antibodies labeled with any of the combinatorial fluorescence energy transfer tags disclosed herein.
The invention provides for the use of any of the combinatorial fluorescence energy transfer tags disclosed herein in gene mutation analysis, in mapping nucleic acids, in chromosome analysis, and in binding assays.
The invention provides a plurality of combinatorial fluorescence energy transfer tags comprising any of the combinatorial fluorescence energy transfer tags disclosed herein, wherein each tag in the plurality of tags has a unique fluorescence emission signature.